The principal objective of this proposal is to gain an understanding of the mechanism by which toxin from the Portuguese Man- of-War (Physalia physalis) affects the cardiovascular system of mammals. Coelenterate toxins appear to contain components which affect impulse transmission within the heart as well as muscle contraction and relaxation directly. Since these components appear to produce various opposing effects under experimental conditions, our initial investigations will emphasize the isolation and purification of those components which affect cardiovascular function. Once purified, these components will be biochemically characterized, their physiological and pharmacological actions evaluated, and a preliminary study of the sub- cellular mechanisms by which they exert their effect undertaken. Those components which interfere with sodium transport across membranes (as measured initially by the short-circuit frog skin apparatus) and those components which interfere with calcium binding to either sarcoplasmic reticulum or mitochondria (as assayed by binding techniques using Ca45 ions) will be isolated using standard protein purification techniques (gel filtration, ion exchange celluloses, ammonium sulfate precipitation, etc). Each isolated component will then be characterized as to molecular weight, isoelectric point, amino acid composition, prosthetic group composition, and possible enzymatic properties. Cardiovascular effects of homogenous fractions will be studied using standard experimental mammals. The ECG, blood pressure, cardiac output and various blood parameters will be analyzed. Myocardial effects will be examined using whole perfused heart preparations, as well as isolated Purkinje fibers. Other muscle effects will be studied using isolated nerve-muscle preparations, as well as both "skinned" and glycerinated muscle. Effects at the level of the cell membrane will be investigated using voltage-clamp techniques, electron microscopy, and appropriate enzyme inhibition studies.